Frizzled6 deficiency disrupts the differentiation process of nail development

Nails protect the soft tissue of the tips of digits in humans. In birds and mammals the equivalent claws are used for purposes including capturing prey, digging, climbing, fighting and maintaining dexterity and balance. The molecular mechanism of nail (and claw) development is largely unknown, but we have recently identified a Wnt receptor gene, Frizzled6 (Fzd6), as mutated in a human autosomal-recessive nail dysplasia. To investigate the action of Fzd6 in claw development at the molecular level, we compared gene expression profiles of digit tips of wild-type and Fzd6-/- mice, and show that Fzd6 regulates the transcription of a striking number of epidermal differentiation-related genes. Sixty-three genes encoding keratins, keratin associated proteins, and transglutaminases and their substrates were significantly down-regulated in the knockout mice. Among them, four hard keratins, Krt86, Krt81, Krt34 and Krt31; two epithelial keratins, Krt6a and Krt6b; and transglutaminase1 were known to be expressed in nails and involved in nail abnormality when dysregulated. Immunohistochemical studies revealed decreased expression of Krt86, Krt6b and involucrin in the epidermal portion of the claw field in the knockout embryos. We further show that Dkk4, a Wnt antagonist, was significantly down-regulated in Fzd6-/- mice along with Wnt, Bmp and Hh family genes; and Dkk4 transgenic mice showed a subtly but appreciably modified claw phenotype. Thus, Fzd6-mediated Wnt signaling likely regulates the overall differentiation process of nail/claw formation. Heterozygous Fzd6+/- mice (kindly provided by Dr. J. Nathans) were crossed to generate Fzd6+/-, Fzd6-/- and wild-type offspring. Timed matings were set up to harvest embryos at E14.5, E16.5 and E18.5. The morning after mating was designated as E0.5. Tails and the digital tips from forelimbs of three wild-type and Fzd6-/- mice from each embryonic time-point were excised, immediately frozen on dry ice, and stored at 80 C freezer until use. Genotyping was done as described previously (15). RNA was extracted using Trizol Reagent (Life technologies). RNA from dissected forelimbs from each embryo of the three time-points was used for microarray analysis.在人类中，指（趾）甲能够保护指（趾）端的软组织。在鸟类与哺乳动物中，与之功能等价的爪则被用于捕猎、挖掘、攀爬、争斗以及维持动作灵活性与身体平衡等多种场景。目前对于甲（及爪）发育的分子机制尚不清楚，但我们近期在一例人类常染色体隐性遗传性甲发育不良患者中，发现了Wnt受体基因Frizzled6（Fzd6）发生了突变。为从分子层面探究Fzd6在爪发育过程中的作用机制，我们对野生型与Fzd6基因敲除（Fzd6-/-）小鼠的指（趾）端组织进行了基因表达谱对比分析，结果显示Fzd6可调控大量表皮分化相关基因的转录。在该基因敲除小鼠体内，共计63个编码角蛋白、角蛋白关联蛋白以及转谷氨酰胺酶及其底物的基因出现了显著的表达下调。其中，4种硬角蛋白Krt86、Krt81、Krt34与Krt31，2种上皮角蛋白Krt6a与Krt6b，以及转谷氨酰胺酶1，均已被证实可在甲组织中表达，且当其表达失调时会引发甲结构异常。免疫组织化学实验结果显示，基因敲除胚胎的爪发育区域表皮部分中，Krt86、Krt6b以及外皮蛋白的表达水平出现了下降。我们进一步研究发现，作为Wnt信号通路拮抗剂的Dkk4，与Wnt、Bmp及Hh家族基因一同在Fzd6-/-小鼠中出现了显著表达下调；同时，Dkk4转基因小鼠的爪表型出现了细微但可被观测到的改变。综上，Fzd6介导的Wnt信号通路很可能调控了甲/爪形成的整体分化过程。我们将由J. Nathans博士惠赠的杂合子Fzd6+/-小鼠进行杂交，以获得Fzd6+/-、Fzd6-/-以及野生型子代小鼠。我们设置了定时交配实验，以分别在胚胎发育第14.5天（E14.5）、第16.5天（E16.5）及第18.5天（E18.5）采集胚胎。交配后的清晨被记为胚胎发育第0.5天（E0.5）。每个胚胎发育时间点的野生型与Fzd6-/-小鼠各3只，我们摘取其尾部及前肢指（趾）端组织，即刻置于干冰上速冻，随后保存于-80℃冰箱中直至后续实验使用。基因分型实验按照此前已发表的方法进行（15）。总RNA提取采用Trizol试剂（Life Technologies公司）完成。我们将三个发育时间点下每个胚胎的解剖前肢组织所提取的RNA，用于基因芯片分析。